The dairy industry represents a major source of income for many people throughout the world, and dairy products represent a significant source of nutrition for a majority of the world's human and non-human animal populations in the form of milk, cheese, yogurt, and other products. In 1994, the U.S. dairy (and egg) industry contributed approximately $717,000,000 to U.S. exports. The import figure for these same commodities was $583,000,000 (R. Famighetti (ed.), World Almanac and Book of Facts, 1996 Funk & Wagnalls, Mahway, N.J., [1995], p. 207).
At least part of the dairy industry's success is due to the increased efficiency in milk production by various cattle breeds, as well as the increased efficiency of milk processing capabilities. Among the beneficiaries of this increased efficiency are the makers of cheese. The ancient art of cheese making represents a significant source of income for many areas, including the "dairy state" of Wisconsin, as well as many other locales. Although there are many variations, the primary steps in cheese making are shown in FIG. 1.
Cheese is made from the milk of various mammals, including cattle, sheep, goats, reindeer, and buffalo. Differences in the texture and tastes of cheeses produced from the milk of these animals are largely due to the differences in the milk compositions. Differences in these constituents also sometime contributes to the inability of animals of one species to utilize the nutrition available in the milk from other animal species. For example, some individuals are completely unable to tolerate cow's milk. These infants are often provided with goat's milk as a substitute formula.
The basic components of milk include fat, protein, lactose, ash, enzymes and vitamins. Table 1 lists the percent solids, and the percent solids comprised of fat, protein, and carbohydrates (R. W. Kirk and S. I. Bistner, Handbook of Veterinary Procedures and Emergency Treatment, 2d ed., W. B. Saunders Co., Philadelphia, Pa. [1975], pp. 644-649).
TABLE 1 ______________________________________ Chemical Composition (%) of Milk % of Solids % Carbo- Animal % Solids % Fat % Protein hydrates % Ash ______________________________________ Cattle 11.9 29.9 25.6 38.7 5.8 Goats 12.8 32.0 29.0 32.8 6.2 Sheep 20.5 41.9 27.9 26.3 3.9 Swine 20.0 36.6 33.0 24.8 5.6 Horses 10.9 14.4 20.2 56.6 8.8 Rhesus 12.2 31.9 17.2 48.4 2.5 Monkeys ______________________________________
There are two major categories of milk proteins. The first is present as a suspension (colloid) known as "casein," while the second form referred to as "whey protein" is soluble. Other compounds present in the protein component of milk include peptones, non-proteinaceous nitrogenous compounds, and various enzymes. When milk is acidified (i.e., becomes sour) in the presence of coagulating enzymes, the casein is altered in such a manner that "curd" is formed. Curd is then used to make cheese. The liquid portion of the soured milk is the "whey." As shown in FIG. 1, whey is a by-product of the cheesemaking process. In the past, the whey was discarded as waste or used for livestock feed, although whey made by acid coagulation and high heat (e.g., during the making of cheddar, Swiss or provolone cheeses) may be used to make ricotta cheese (A. M. Pearl et al., Completely Cheese: The Cheeselover's Companion, Jonathan David Publishers, Inc., Middle Village, N.Y., [1978], p. 59-60). Other cheeses made principally from whey include such Scandinavian cheeses as Gjetost, made from goat's milk whey, and Mysost, made from cow's milk whey, as well as Sapsago, a cheese made in Switzerland from cow's milk whey. Nonetheless, in the past whey was primarily considered to be a waste product of cheese-making.
In the cheese industry, two types of precipitation techniques are most commonly used to separate the total milk proteins separated into caseins and whey proteins--rennet precipitation and acid precipitation. In rennet precipitation, rennin is added to warm milk (30-35.degree. C.), precipitating the caseins and leaving the whey proteins in solution. Whey produced by this method is referred to as "sweet whey." Acid precipitation is carried out at the isoelectric point of milk (i.e., 4.7) through the use of acid to precipitate out casein and leave the whey in solution. Whey produced by this method is referred to as "acid whey." The choice of the method used to make the curd depends on the desired cheese product.
Although the whey is discarded during the cheesemaking process, it has significant value as a source of nutrition. Unless whey is used to produce other products (including feed), it is a liability for cheese factories. The high biological oxygen demand (BOD) of whey presents disposal problems. Thus, large quantities of whey are used in candies and special cheese products. In addition, large quantities of dry and concentrated forms of whey are also used in dry and concentrated forms that are mixed with food and feeds. Whey may also be condensed and spray dried by the roller process for other uses. Various whey drinks have been developed, including wines, carbonated beverages, buttermilk substitutes, whey-fruit flavored drinks, and whey-tomato drinks. Whey is also used to produce whey butter, soups, protein hydrolysates, cheeses, processed cheese foods and spreads, bakery products, infant foods, geriatric foods, hydrolyzed lactose syrup, pills, riboflavin concentrates, alcohol (e.g., butyl alcohol), methane, acetone, spirit vinegar, food acidulant, resins, coatings, tanning, acrylic plastics, and biomass (See e.g., Frandsen (ed.), Dairy Handbook and Dictionary, J. H. Frandsen, Amherst, Mass. [1958], at pages 791-792; and Taylor, Scientific Farm Animal Production (5th ed.), Prentice Hall, Englewood Cliffs, N.J., [1995], p. 93). In their undenatured, soluble form, whey proteins are also useful as binders in extruded vegetable or animal protein foods (See e.g., Rosenthal, Milk and Dairy Products, VCH, New York, N.Y., [1991], at p. 137).
Whey contains various proteins (e.g., .beta.-lactoglobulin and .alpha.-lactalbumin), lactose, soluble minerals, water-soluble vitamins, and enzymes. Protein compounds present in whey have received the most attention, for their potential utilization in various foods, feeds, and other products as mentioned above. Thus, separation of these compounds has been studied. The following table lists some of the properties and concentrations of these compounds.
TABLE 2 ______________________________________ MOLECULAR WEIGHTS OF SELECTED WHEY PROTEINS Approx. Isoelectric Concentration in Protein Point Molecular Weight Whey (g/l) ______________________________________ .beta.-Lactoglobulin 5.35-5.49 18,300 3.0 .alpha.-Lactalbumin 4.2-4.5 14,000 0.7 Immunoglobulins 5.5-8.3 .gtoreq.150 000 0.6 Bovine Serum 5.13 69,000 0.3 Albumin Protease-Peptones 3.3-3.7 4,100-40,800 1.4 Lactoferrin 7.8-8.0 77,000 0.03 Lactoperoxidase 9.2-9.9 77,500 0.02 ______________________________________
Indeed, various methods are commercially available for the separation, removal, concentration, and/or purification of selected whey proteins, including the methods of such publications as: U.S. Pat. No. 5,077,067, which discloses a process for the selective and quantitative removal of lactoglobulins from whey proteins; U.S. Pat. No. 5,055,558, which describes a method for the selective extraction of .beta.-lactoglobulin from whey or milk by subunit exchange chromatography; U.S. Pat. No. 4,791,193, which discloses a method for the preparation of pure lactoferrin from whey or skim milk; U.S. Pat. No. 4,668,771, which describes a method for the isolation and purification of bovine lactoferrin; U.S. Pat. No. 4,997,914 which describes an adsorption chromatography method for the separation and purification of lactoferrin; U.S. Pat. No. 4,820,348, which directed to a chromatographic method for the separation of lactose from milk; U.S. Pat. No. 4,446,164, which discloses milk-like compositions constituted from a sweet whey base with additives such as soluble proteins, edible vegetable oils, non-fat dry milk solids, sugar or synthetic sweeteners; U.S. Pat. No. 5,085,881, which describes a process for separating fractions from dried milk or milk products for use as food stuffs or food or pharmaceutical adjuvants; U.S. Pat. No. 5,093,143, which discloses nutrient compositions that simulate milk and are rich in energy and calcium content but poor in albumin and phosphorus; U.S. Patent No. 4,202,909, which describes a process for the treatment of whey to produce pure lactose and salt products; U.S. Pat. No. 5,008,376, which discloses a process for producing a whey fraction with a high concentration of .alpha.-lactalbumin by ultrafiltration technology, and U.S. Pat. No. 3,969,337, which discloses a method for the chromatographic fractionation of whey. However, all of these methods have significant disadvantages, including the fact that all result in the destruction or disposal of all but one selected protein from the whey, thereby wasting the other valuable proteins obtained during the separation process.
Furthermore, the cheese industry produces large amounts of whey, much of which is used to make whey protein concentrate (WPC). WPC is mainly produced by the thermocalcic pretreatment, ultrafiltration (UF) and microfiltration (MF) of whey. However, WPC has some undesirable properties, such as low foam formation, and poor foam stability due to its high lactose and lipid content (Karleskind, et. al. 1995). These undesirable properties are mainly eliminated for whey protein isolate (WPI), which is made by adsorption of proteins directly from whey onto ion-exchange beads. In commercial WPI manufacturing, whey proteins are adsorbed into ion-exchange beads, followed by washing, elution of the adsorbed protein, cleaning, and regeneration of the beads. The rate of WPI production or fractionation of whey proteins using conventional ion-exchange beads are slow (See, Wit, et al. 1995, Uchida et al. 1993, and de Rahm el al. 1989).
Commercial-scale fractionation of different whey proteins has been hampered by the lack of an economical fractionation technology. The resolution and throughput of conventional chromatographic methods such as stirred tanks and packed axial columns is too low to be economical. This is especially true because the whey proteins are present in small quantities. Thus, in order to recover a fixed amount of protein, large volumes of solution must be processed.
In addition, conventional ion-exchange beads have limitations. For example, the equilibrium rate is slow in large ion-exchange beads due to the lengthy times required for diffusion of the proteins into the beads. Smaller ion-exchange beads decrease the diffusion time, but this causes an increase in the liquid drainage time through the bed. Consequently, the throughput of ion-exchange process is limited by either slow intra-bead diffusion for large beads or slow liquid drainage rate and high column pressure drops (i.e., in packed bed columns) for small beads.
Several regenerated cellulose ion-exchange beads are also available for whey protein isolation. However, these cellulosic ion exchange beads suffer from the disadvantages of low protein capacity and high price. These two factors have greatly hindered the commercial application of these beads for use with whey (Ayers and Petersen, 1985).
None of these methods achieve the separation of various proteins from whey in a single process step. It would be desirable, therefore, to provide a method for the continuous and sequential separation of various proteins from whey in a simple one or two step separation process. Furthermore, none of these methods provide a product which can be easily, economically, and efficiently utilized as a supplement for infant formulas, fat substitutes or other commercially important products.
The cost of producing WPI is higher than WPC, primarily due to higher capital costs for building the ion-exchange plant compared to the UF and MF plant (Etzel, 1995). In order to market WPI at an economically reasonable lower cost, either the process efficiency and throughput must be increased or the capital cost must be decreased. Thus, a need remains in the industry for economical methods to process whey products, including processes and methods that are designed so as to permit reutilization of buffers and constituents utilized during the purification of whey compounds. In addition, methods are needed to increase the efficiency of whey processing procedures, including methods designed to reduce the time necessary to obtain a final product.